1. A Ligand-Activated Nuclear Localization Signal in Cellular Retinoic Acid Binding Protein- II 
Richard J. Sessler, Noa Noy 
Molecular Cell 
Volume 18, Issue 3, Pages (April 2005)
DOI: /j.molcel
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2. Figure 1 CRABP-II Localizes to the Nucleus in Response to RA
MCF-7 cells were treated with ethanol (A–C) or 100 nM RA (D–F) for 30 min. (A and D) Immunostaining with CRABP-II antibodies; (B and E) Imaging of nuclei using the nucleic acid stain ToPro3; (C and F) Overlay of images of CRABP-II and nuclei.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 RA Induces Nuclear Import of GFP-CRABP-II
COS-7 cells were transfected with expression vectors harboring the denoted CRABP-II constructs. Images of GFP-tagged proteins were acquired from live cells before (A, C, E, G, and I) or after (B, D, F, H, and J) a 30 min treatment with RA (1 μM). Treatment with leptomycin B (LMB, 5 ng/ml) was carried out for 5 hr prior to and throughout imaging.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Structural Features of CRABP-II and Alignment of Holo-CRABP-II-K20/R29/K30 with the NLS of the SV40-T Antigen
(A) Superposition of the of apo- (blue) (Chen et al., 1998) and holo-CRABP-II (green) (Kleywegt et al., 1994). Bound RA is shown in gray.
(B) Computed electrostatic surface potentials of apo- and holo-CRABP-II (see Experimental Procedures). Basic, acidic, and neutral charges are denoted by blue, red, and white, respectively. A positively charged patch (arrow) is manifested in holo-CRABP-II.
(C) Structures of helix 2 of apo- and holo-CRABP-II showing the region that tightens in response to RA binding (arrows).
(D) Structures of the helix-loop-helix regions of apo- and holo-CRABP-II showing that three basic residues, K20, R29, and K30, change positions upon ligation.
(E) Structures of helix 1 showing increased distance between E17 and K20 in the holoprotein.
(F) Structure of the SV40 NLS peptide bound to karyopherinα (Conti et al., 1998). K128, K129, and K131 (blue) comprise the consensus NLS of SV40.
(G) Superposition of residues K20, R29, and K30 of holo-CRABP-II (green) with K128, K129, and K131 of the SV40 NLS peptide (blue).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
RA Induces Association of CRABP-II with Importin α and Mutations of K20, R29, and K30 Hinder the Interactions
(A) SDS-PAGE of bacterially expressed wild-type (WT)-CRABP-II and its mutant (KRK/AAA).
(B) Urea-induced unfolding of CRABP-II (squares) and mutant (circles). Inset: fluorescence emission spectra of WT-CRABP-II (λex = 283 nm) in the absence (solid line) or presence (dotted line) of 9 M urea. Urea-induced shift in emission spectra is presented as the ratio of fluorescence intensities at 343 nm and 357 nm.
(C) Representative fluorescence titrations of WT- and mutant CRABP-II. Data were fitted to an equation derived from simple binding theory (solid line through data points).
(D) GST pull-down experiments using 35S-labeled importin α/hSRP1 and GST, or GST-tagged CRABP-II, or its mutant (KRK/AAA), in the presence or absence of RA (1.5 μM). 35S- importin α and bait proteins were visualized by autoradiography and by Coomassie blue staining, respectively. The experiment was carried out three times with similar results.
(E) Quantitation of bands in (D). Intensities were measured using Alpha Innotech AlphaEase version 5.5 software for image capture and densitometry and were normalized to intensity observed with unliganded WT-CRABP-II.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Mutation of the Putative NLS Abolishes Ligand-Induced Nuclear Localization of CRABP-II
COS-7 cells were transfected with GFP-WT-CRABP-II or its K20A/R29A/K30A mutant (KRK/AAA). Images of GFP-tagged proteins (green) and of the nuclear marker Red1-nuc (blue) were acquired from live cells before or after a 30 min treatment with RA (1 μM).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Nuclear Localization Is Required for CRABP-II-Mediated Enhancement of the Transcriptional Activity of RAR
COS-7 cells were transfected with a luciferase reporter driven by an RAR response element, a pCH110 vector (internal standard) and either an empty vector (pSG5), or expression vectors for the denoted CRABP-II constructs. Cells were treated with RA (250 nM) or vehicle overnight prior to measurements of luciferase activity. Luciferase activity was normalized to β-gal activity and fold activation is presented. Data are means ± standard deviation (n = 9).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Alignment of iLBPs with the NLS-Containing Region of CRABP-II
Some iLBPs contain residues that correspond in charge to the CRABP-II-NLS (underlined), while others do not (bold). Alignment was accomplished using accession numbers: NP_ (hCRABP-II), P15090 (hA-FABP), NP_ (hK-FABP), NP_ (hCRABP-I), NP_ (hCRBP-I), NP_ (hCRBP-II), NP_ (hL-FABP), P12104 (hI-FABP), and P05413 (hH-FABP).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions